Use of DACEE and Derivatives for treating viral diseases

ABSTRACT

The present invention relates to a new use of N,S-diacetylcysteine ethyl ester for the preparation of a pharmaceutical composition for treating virus-induced disease. In particular, the invention concerns the use of DACEE for the preparation of a pharmaceutical composition for treating virus-induced disease, the DACEE used destroying disulfide bridges present in viral proteins.

This is a national phase filing of the Application No. PCT/EP94/02505,which was filed with the Patent Cooperation Treaty on Jul. 28, 1994, andis entitled to priority of the German Patent Application No. P 43 25547.7, filed Jul. 29, 1993.

I. FIELD OF THE INVENTION

The present invention relates to a new use of thiol compounds for thepreparation of a pharmaceutical composition for treating virus-modifieddiseases. In particular, this invention concerns a use of thiolcompounds for the preparation of a pharmaceutical composition fortreating virus-induced disease, the thiol compounds used destroyingdisulfide bridges present in proteins.

II. BACKGROUND OF THE INVENTION

Acute and chronic infections of plants, animals and man, caused byvarious viral pathogens, represent a serious medical and economicproblem. In fact, presumably 60% of the diseases occurring in theindustrialized countries are caused by viruses. Thus, diseases caused byhepatitis viruses, for example, are counted among the most frequentinfections world-wide. Since said viral infections substantially affectthe liver, the progressive destruction of this organ, followed by asubsequent development of a cirrhosis, may finally result in theformation of a hepatocellular carcinoma.

A high specificity degree for the viral pathogen and the simultaneousabsence or minimization of health-damaging side-effects have to beregarded as an essential demand to be made on an antiviral therapeuticagent. In this connection, the close linkage of the viral reproductivecycle with the metabolic and replicative functions of the host cellprove to be especially problematic. For the time being, medical researchfocuses on the development of antiviral agents which impair or preventthe replication of the viral genome, great importance being attachedparticularly to chemically synthesized nucleoside analogues.

However, one of the main problems of antiviral chemotherapy has to beseen in the fact that no effective substances for therapeutic treatmentare available for plurality of significant infective agents such as thehepatitis B virus, for example.

Correspondingly, the present invention is based on the technical problemof providing further therapeutically active and simultaneouslypharmaceutically compatible substances for controlling viral diseases.

The solution to this technical is achieved by providing the embodimentscharacterized in more detail in the claims. In particular, the technicalproblem is solved in that the present invention discloses thetherapeutically active use of thiol compounds for the preparation of apharmaceutical composition for controlling virus-induced diseases,disulfide bridges present in viral proteins being destroyed by the thiolcompounds.

III. SUMMARY OF THE INVENTION

The present invention relates to the new use of thiol compounds for thepreparation of a pharmaceutical composition for treating virus-induceddisease. In particular, the invention concerns the use of thiolcompounds for the preparation of a pharmaceutical composition fortreating virus-induced disease, the thiol compounds used destroyingdisulfide bridges present in viral proteins.

IV. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the content, i.e. the amount detected via immunologicalinteraction between viral surface antigen and commercially obtainableantibody, of the viral antigen HBsAG in the cell culture supernatant ofHepG2-4A5 cells after treatment with various NAC concentrations as afunction of time.

FIG. 2 shows the content of the viral antigen HBeAg in the cell culturesupernatant of HepG2-4A5 cells after treatment with various NACconcentrations as a function of time.

FIG. 3A shows the content of intact HBV viral particles in the cellculture supernatant of HepG2-4A5, the cell line HepG2-4A5 having beenincubated additionally with 30 nM NAC concentrations. As a control, thecell supernatant of the cell line HepG2 is shown which does not produceHBV particles (ATCC HB 8065). See, Nature, 1979, 282:615-616.

FIG. 3B shows the content of intact HBV viral particles in the cellculture supernatant of HepG2-4A5having been incubated with varying NACconcentrations. As a control the cell supernatant of the cell line HepG2is shown which does not produce HBV particles (ATCC HB 8065). See,Nature, 1979, 282:615-616, supra.

FIG. 4A shows the content of HBsAg and HBeAg in a purchasableHBsAg-positive serum, the serum having been incubated with varyingconcentrations of NAC.

FIG. 4B shows the content of HBsAg and HBeAg in a purchasableHBsAg-positive serum, the serum having been incubated with varyingconcentrations of cysteine.

FIG. 4C shows the content of HBsAg and HBeAg in a purchasableHBsAg-positive serum, the serum having been incubated with varyingconcentrations of DACEE.

FIG. 5 shows the content of infections vaccinia virus particles, basedon the plaque number in the cell culture monolayer of CV1 cells infectedwith vaccinia virus (ATCC CCL70), the viruses having been preincubatedwith varying NAC concentrations prior to the infection.

V. DETAILED DESCRIPTION OF THE INVENTION

The expression "thiol compound" is understood to mean chemical compoundswhich are characterized by the presence of a reduced thiol group (SHgroup), as well as compounds which in the course of the cellularmetabolism can be converted into thiol compounds. Preferred examples ofsuch thiol compounds are cysteine, cysteine derivatives,mercaptoalkanols, such as methanethiol, ethanethiol or mercaptoethanol,dithiocarbamate, thiophenol and 2-mercaptoetthanesulfonic acid.N-acetylcysteine derivatives such as N-acetylhomocysteine,N-acetylcysteine ethyl ester or N,S-diacetylcysteine ethyl ester areexamples of cysteine derivatives according to the invention.N-acetylcysteine (NAC) and N,S-diacetylcysteine ethyl ester (DACEE) aswell as the derivatives thereof are especially preferred. The thiolcompounds used according to the invention are also characterized bytheir non-toxicity within the concentration range usually used fortherapeutic treatments (in the case of NAC: e.g., 600 mg daily overseveral weeks). In the medical field, NAC is presently used as amucolytic agent, in the case of paracetamol intoxication andexperimentally as HIV and herpes therapeutic agent. Roederer et al.,1990, Proc. Natl. Acad. Sci. U.S.A. 87:4884-4888; U.S. Pat. No. 4 708965. The thiol compounds according to the invention are characterizedparticularly by a good compatibility and minor side-effects. Thus, theydistinguish themselves particularly from the nucleoside analogues usedfor the virus therapy up to now.

The expression "virus-induced disease" is understood to mead diseases ofthe animal and human bodies, which are caused or intensified by viruses.According to the invention they include particularly DNA viruses such ashepadnaviridae, adenoviridae, parvoviridae, papovariridae, adenoviridae,poxviridae, iridoviridae, or RNA viruses, such as picornaviridae,calciviridae, togaviridae, flaviviridae, coronaviridae, rhabdoviridae,filoviridae, paramyxoviridae, orthomyxoviridae, bunyaviridae,arenaviridae, reoviridae, birnaviridae. Within the meaning of thisdefinition herpes viruses and AIDS viruses are not considered to be RNAviruses. preferably to the use of thiol compounds for controllingvirus-induced diseases which are caused by hepatitis viruses such ashepatitis A, B, C or delta virus. In particular, the virus-induceddisease may be a chronic or acute hepatitis B infection or ahepatocellular carcinoma. Particularly, the expression "virus-induceddiseases" is understood to mean diseases which are caused by hepadnaviruses such as the hepatitis B virus, the "Woodchuck Hepatitis Virus"(WHV), the "Ground Squirrel Hepatitis Virus" (GSHV), the "Tree SquirrelHepatitis Virus" (TSHV) or the "Duck Hepatitis Virus" (DHBV), forexample. Furthermore, this invention also comprises the use of thiolcompounds for controlling vegetable disease which are caused by viralpathogens such as the tobacco mosaic virus (TMV) or the cauliflowermosaic virus (CaMV).

The expression "disulfide bridges present in viral proteins" refers todisulfide bridges which influence the spatial structure and/or thefunction of viral proteins by intramolecular or intermolecular covalentbonds. Correspondingly, the viral proteins, but at least one viralprotein, of the use of thiol compounds according to the inventioncontain at least one cystein residue. Preferably the viral proteincontains several cysteine residues such as HBsAG of the hepatitis Bvirus, for example. It is well known that disulfide bridges arestabilized by the conformation of the protein, on the other hand. Thus,the amount of thiol compound to be used, which is necessary for thecleavage of the intramolecular or intermolecular disulfide bridges,depends on the accessibility of the respective disulfide bridge(s).

The expression "viral proteins" is understood to mean proteins,preferably coat proteins, such as HBcAG and HBsAG, of a virus. Inparticular, this expression refers to surface antigens proteins such asHBsAG, for example.

According to the invention, the expression "viral proteins" also includeprecursor proteins for coat proteins, i.e. proteins which are notpresent in the final virus.

Furthermore, this invention relates to the use of thiol compounds forthe preparation of a pharmaceutical composition for treatingvirus-induced disease, disulfide bridges present in viral proteins beingdestroyed by the thiol compound and the thiol compound not influencingthe replication of the viral genome.

The following preparations and examples are given to enable thoseskilled in the art to more clearly understand and to practice thepresent invention. The present invention is not limited in scope by theexemplified embodiments, which are intended as illustrations of singleaspects of the invention only, and methods which are functionallyequivalent are within the scope of the invention. Indeed, variousmodifications of the invention in addition to those described hereinwill become apparent to those skilled in the art from the foregoingdescription and accompanying drawings. Such modifications are intendedto fall within the scope of the appended claims. The present inventionis explained in more detail by means of the below examples.

VI. EXAMPLES A. Example 1 Influence Of The Thiol Compounds on theReplication of the Hepatitis B Virus

In order to investigate the influence of thiol compounds on thereplication of the hepatitis B virus, use was made of the stablytransfected, potentially infectious HBV particle-producing cell linesHepG2 2.2.15 (Sells et al., 1987, Proc. Natl. Acad. Sci. U.S.A.84:1005-1009) and HepG2.4A5 (HepG2 cell line stably transfected with theplasmid pSPT1, 2XHBVneo (cf. Example 4)). NAC, cystein, cysteinhydrochloride and DACEE were used as thiol compounds. Cystein wasemployed s the negative control.

TABLE I shows the HBsAg and HBeAg contents of the cell culturesupernatant of the HepG2-4A5 cell line 48 hours after treating the cellswith the above compounds. In this connection, the HBsAg content andHBeAg content, respectively, of the untreated cells was arbitrarily setto 1 and the inhibitory index said compounds was determined in relationthereto. Just as in Examples 2 and 3, the antigens were determined bymeans of a commercially available HbsAg enzyme immunoassay and HBeAgenzyme immunoassay, respectively (Abbott-Laboratories).

The results shown in TABLE I were obtained for NAC also with the cellline HepG2 2.2.15 and, after transient transfection of HepG2 cells (andHuH7 cells, respectively), with the replication-competent HBV plasmidpSPT1, 2xHBV. pSPT1, 2xHBV was pared as described in Example 4.

                  TABLE I                                                         ______________________________________                                        SUBSTANCE                                                                              CONC.      HBsAg      HBeAg                                          ______________________________________                                        untreated           0.189 ± 0.016                                                                         0.386 ± 0.048                               NAC       3 mM      0.085 ± 0.011                                                                         0.323 ± 0.029                                        10 mM      0.023 ± 0.002                                                                         0.295 ± 0.034                                        30 mM      0.003 ± 0.001                                                                          0.113 ± 0.0121                             cysteine  3 mM      0.154 ± 0.039                                                                         0.348 ± 0.039                                        10 mM      0.129 ± 0.024                                                                         0.302 ± 0.029                                        30 mM      0.053 ± 0.006                                                                         0.084 ± 0.006                               μg/ml DACEE                                                                          0         0.241 ± 0.056                                                                         0.310 ± 0.009                                           0.05    0.173 ± 0.034                                                                         0.281 ± 0.021                                           0.5     0.197 ± 0.031                                                                         0.283 ± 0.004                                         5         0.226 ± 0.023                                                                         0.318 ± 0.009                                         10        0.206 ± 0.018                                                                         0.302 ± 0.026                                         20        0.206 ± 0.004                                                                         0.332 ± 0.019                                         50        0.157 ± 0.009                                                                         0.323 ± 0.013                                        100        0.079 ± 0.006                                                                         0.267 ± 0.007                                        150        0.077 ± 0.009                                                                         0.249 ± 0.013                                        300        0.004 ± 0.002                                                                         0.211 ± 0.007                                        600        0.002 ± 0.001                                                                         0.179 ± 0.004                                        2000       0.002 ± 0.001                                                                         0.139 ± 0.009                               ______________________________________                                    

A marked reduction of HBsAg in the cell culture supernatant could beproved from both NAC-treated cells as well as cysteine- andDACEE-treated cells. A diagram of the above data is depicted in FIGS.4A, 4B, and 4C. As compared thereto, the control compound cystine showedno influence on the content of the secretable viral antigens, HBsAg andHBeAg (data not shown).

Furthermore, the dose-dependent, inhibitory effect of NAC on the contentof viral antigens in the cell culture supernatant was investigated bymeans of the above HBsAg enzyme immunoassay and HBeAg enzymeimmunoassay, respectively, as a function of the incubation period (FIGS.1 and 2). The NAC concentrations used in this connection show nocytotoxic effect on the cells of the cell culture used. The dataillustrated in the figure shows a marked dose-dependent inhibitoryeffect on the content of the investigated viral antigens in the cellculture supernatant.

In order to show that the content of intact and thus potentiallyinfectious viral particles in the cell culture supernatant of theHepG2-4A5 cell line is reduced after treating the cells with thiolcompounds, an endogenous-polymerase reaction was carried out. Thisreaction profits from the fact that the viral polymerase present in theviral capsid can complete in vitro the partially double-stranded HBVgenome. By the addition of radioactively labelled nucleotides andsubsequent incorporation into the HBV genome it is thus possible toprove the presence of intact, potentially infectious viral particles ina cell culture supernatant. The results shown in FIG. 3A show clearlythat the content of potentially infectious intact viral particles in thecell culture supernatant is markedly reduced after treating the cellswith NAC.

This result was confirmed in another experiment illustrated in FIG. 3B.For this purpose, the viral particles in the cell culture supernatant ofthe cell line HepG2-4A5 treated with varying NAC concentrations werespecifically enriched by immunoprecipitation with a HBcAg antibody (DAKOGmbH, Hamburg), after removing their outer coat, and then anendogenous-polymer reaction was carried out. Here, too a marked decreaseof the hepatitis B virions in the cell culture supernatant occurs aftertreatment with NAC.

B. Example 2 Investigations Made as to the Mode of Action of NAC,Cysteine and DACEE

In order to investigate whether the antiviral mechanism of action ofNAC, cysteine and DACEE, respectively, is based on the destruction ofthe disulfide bridges present in the viral protein, the cell culturesupernatant of the HepG2-4A5 cell line was incubated with the NACconcentrations indicated in TABLE II overnight and then the HBsAgcontent was determined by means of a purchasable test method(Abbott-Laboratories).

Likewise, purchasable HBsAg-positive serum (Abbott-Laboratories) waspreincubated with the NAC, cysteine, DACEE and cysteine hydrochlorideconcentrations indicated in TABLE III for 5 minutes and then the contentof HBsAg and HBeAg was determined by means of the above test method.

TABLES II and III show a marked decrease of HbsAg as a function of theemployed concentrations of NAC, DACEE and cysteine hydrochloride,respectively. It can be inferred from the entirety of this data that theantiviral effect of NAC, cysteine, DACEE and cysteine hydrochloride,respectively, is based on a reduction of the disulfide bridges in theHBsAg complex of the hepatitis B virus and thus the build-up of thehepatitis B viral coat is disturbed.

                  TABLE II                                                        ______________________________________                                                     HBsAg                                                            ______________________________________                                        untreated      1                                                               3 mM NAC      0.95 ± 0.16                                                 10 mM NAC      0.54 ± 0.04                                                 30 mM NAC      0.22 ± 0.05                                                 100 mM NAC     0.03 ± 0.03                                                 ______________________________________                                    

                  TABLE III                                                       ______________________________________                                        SUBSTANCE CONC.      HBsAg      HBeAg                                         ______________________________________                                        untreated                1.00 ± 0.05                                                                          1.00 ± 01.07                            NAC       3     mM       0.79 ± 0.05                                                                         1.09 ± 0.04                                        10    mM       0.49 ± 0.03                                                                         1.17 ± 0.04                                        30    mM       0.02 ± 0.00                                                                         1.27 ± 0.09                                                       0.91 ± 0.02                                                                         1.10 ± 0.06                              cysteine  3     mM                                                                      10    mM       0.68 ± 0.04                                                                         1.22 ± 0.05                                        30    mM       0.27 ± 0.05                                                                         1.21 ± 0.06                              DACEE     0.6   mg/ml    0.35 ± 0.06                                                                         1.11 ± 0.04                                        2     mg/ml    0.06 ± 0.00                                                                         1.19 ± 0.04                                        6     mg/ml    0.00 ± 0.00                                                                         1.09 ± 0.03                              cysteine  3     mM                                                            hydrochloride                                                                           10    mM        0.84 ± 0.004                                                                       1.17 ± 0.04                                        30    mM       0.33 ± 0.01                                                                         1.32 ± 0.05                                                       0.11 ± 0.01                                                                         1.30 ± 0.08                              ______________________________________                                    

C. Example 3 Influence of NAC on the HBsAg Content of the Human Serum ofan Acutely Infected Patient

In order to investigate whether the in vitro data shown in Example 2 canalso be reproduced by means of human serum, the serum sample of an HBVpatient in the acute stage of infection was incubated with an increasingamount of NAC. In analogy to the in vitro finding, here, too, a markeddecrease of the HBsAg content resulted as a function of the NACconcentration used (TABLE IV).

Since the results shown in TABLE IV were obtained by means of highlyinfectious human serum which has very high HBsAg and HBeAg titers, thesample was diluted to 1:200 prior to the test so as to remain within thelinear range of the HBsAg test used. Preincubation of thisrepresentative experiment was 30 minutes.

TABLE IV shows that--as happens after the incubation of the cell culturesupernatant with NAC--the HBsAg content is markedly also reduced in thehuman serum.

                  TABLE IV                                                        ______________________________________                                                     HBsAg                                                            ______________________________________                                        untreated      1                                                               3 mM NAC      0.27 ± 0.07                                                 10 mM NAC      0.12 ± 0.05                                                 30 mM NAC      0.04 ± 0.00                                                 100 mM NAC     0.00 ± 0.01                                                 ______________________________________                                    

D. Example 4 Preparation of the pSPT1, 2xHBV and pSPT1, 2xHBVneoPlasmids

For preparing pSPT1, 2xHBV, an HBV portion originating from the plasmidpBRHBadr4 (Fujiama et al., NAR 11, 1983, 4601-4610) was cloned into thecloning vector pSPT19 linearized by BamHI (Boehringer Mannheim, orderNo.: 909815). Correspondingly, the plasmid pSPT1, 2xHBV contains an HBVgenome of full length (BamHI fragment) and additionally a terminalredundance of 621 bp (BamHI/Stul fragment). Thus, the plasmid pSPT1,2xHBV contains the minimum portion absolutely necessary to generate allof the viral transcripts, inclusive of the pregonomic RNA having alength of 3.5 kb. This HBV portion is exclusively under the control ofautologous promoters.

For the preparation of pSPT1, 2xHBVneo, a neomycin resistance gene whichis under the control of the herpes simplex TK promoter was additionallycloned into the vector pSPT1, 2xHBV (meomycin portion of PNEO,Pharmacia, order No.: 27-4924-01). In this case, the cloning strategywas chosen such that the HBV portion and the neomycin resistance portion3"-3' face each other so as to keep the influence of the TK promoter onthe HBV portion as low as possible.

E. Example 5 Influence of NAC on the Replication of the Vaccinia Virus

In order to investigate the influence of thiol compounds, e.g., NAC, onthe replication of the vaccinia virus, vaccinia viruses were initiallypreincubated with 3, 10 and 30 mM, respectively, of NAC for 45 minutes.Then, these viruses were incubated with CV1 cells (generally obtainablemonkey kidney cells) for 45 minutes to enable viral adsorption to thecells. Having washed the cells several times, they were covered withsoftager. After an incubation period of about 48 hours, the non-lyzedcells were dyed by means of crystal violet and the resulting plaqueswere counted. The plaque number of the untreated control was set to 1.0and the inhibitory index of the treated cells was determined in relationthereto (cf. TABLE V).

TABLE V discloses that also in the case of the vaccinia virus areduction of the infectious viral particles is achieved by a thiolcompound such as NAC. A diagram of this result is depicted in FIG. 5.

                  TABLE V                                                         ______________________________________                                               mM NAC VACCINA                                                         ______________________________________                                                0     1.00                                                                    3     0.89 ± 0.01                                                         10     0.64 ± 0.07                                                         30     0.33 ± 0.16                                                  ______________________________________                                    

All references cited within the body of the instant specification arehereby incorporated by reference in their entirety.

What is claimed is:
 1. A method for inhibiting propagation of a hepatitis virus, comprising administering N,S-diacetylcysteine ethyl ester (DACEE) in a pharmaceutically acceptable carrier to a subject infected by a hepatitis virus, in an amount sufficient to effect inhibition of hepatitis virus propagation.
 2. The method of claim 1, wherein the virus is a hepatitis B virus.
 3. The method of claim 1, wherein the virus is a hepatitis delta virus.
 4. A method for inhibiting propagation of a hepadna virus, comprising administering DACEE in a pharmaceutically acceptable carrier to a subject infected by a hepadna virus, in an amount sufficient to effect inhibition of hepadna virus propagation. 